Enzyme reactions employing PLP are widely known and utilized. However, PLP is generally employed in an aqueous solution containing the substrate. In such a system, the recovery of PLP after completion of the reaction is extremely difficult. In addition, it is difficult to separate and purify the reaction product in such a system. Further, PLP is not a stable enzyme. Thus, when PLP is employed at neutral pH, room temperature and under normal pressure, the recovery of such is markedly lost. It is thus desired that PLP be stable at wide temperature ranges, at wide pH ranges, and in various solvents and can be recovered easily and stably.
PLP is an exo-enzyme produced by Achromobacter lyticus M 497-1 separated from soil, found by Soejima and Masaki et al deposited in the Foundation, Institute for Fermentation, Osaka under IFO 12725, American Type Culture Collection under ATCC 21456, and Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry of Japan under FERM-P 4420, and is the same enzyme as Achromobacter protease I described in Agricultural and Biological Chemistry, 42:1442 (1978) and Japanese Patent Application (OPI) No. 119085/79.
PLP specifically hydrolyzes the ester bond and the amide bond at the carboxyl group of L-lysine and therefore is useful for semi-synthesis of human insulin from porcine insulin.
It is well known that the optimum temperature of the enzymatic activity of PLP is about 45.degree. C. When casein is used as a substrate, the optimum pH of enzymatic activity is between 8.5 and 10.5. The molecular weight thereof determined by gel filtration is 27,000. The isoelectric point of PLP is pH 6.9. PLP is a serine enzyme that is strongly inhibited by diisopropyl phosphofluoride and tosyl-L-lysine chloromethyl ketone but is not inhibited by tosyl-L-phenylalanine chloromethyl ketone, ethylene-diaminetetraacetate, orthophenanthroline or p-chloromercury benzoate.
The conventional semi-synthesis of human insulin from porcine insulin involves a separation step and requires much time since PLP is used in an aqueous reaction in the form of its monomer and no contamination of human insulin is permitted. In addition, during the separation step, PLP is inactivated in this semi-synthesis and thus it is impossible to reuse the recovered PLP. This is a serious economical drawback in the use of PLP monomers.
Enzyme immobilization has been employed with PLP, however, immobilized PLP becomes insoluble and thus the enzyme reaction must be carried out in a suspension state, etc. Hence, special devices and skill are required for a smooth reaction. That is, a device for effective stirring to obtain a uniform suspension state and a column for prolonging the contact time are necessary. In addition, the use of a relatively large amount of a suitable organic solvent in combination with the use of a large excess of enzyme is disadvantageously required in such a system.